Chemokine receptors (CRs) have drawn much attention since their description as human immunodeficiency virus (HIV) co-receptors by several groups in 1996. Prior to that time, HIV tropism was defined as either macrophage (M)- or T cell (T)-tropic, which corresponded to non-syncytia- or syncytia-inducing viruses, respectively. Today, the classification of HIV tropism is defined by chemokine receptor usage of either CCR5, CXCR4, or both receptors. Chemokine receptors are a family of seven transmembrane spanning G protein-coupled receptors (GPCRs) that are differentially expressed by a number of immune and non-immune cell populations. CCR5 has been shown to be palmitoylated and targeted to cholesterol-and sphingolipid-rich membrane microdomains termed lipid rafts. Lipid rafts is a broad term for the collection of membrane microdomains enriched in cholesterol, sphingolipids, glycosylphosphatidylinositol (GPI)-anchored proteins, and acylated signaling molecules. Lipid rafts are believed to be important signaling platforms enriched in many signaling proteins, including but not limited to src kinases, Ga subunit, H-Ras, LAT, and NOS. Signal transduction through the T and B cell receptors as well as the IgE receptor involves the recruitment of signaling assemblies to lipid rafts. CCR5 has been shown to be present in lipid rafts, colocalizing at the leading edge of migrating cells. This receptor has also recently been shown to be palmitoylated which is one of the important modifications in lipid raft targeting of proteins. However, the role of cholesterol and these lipid rafts on T cell chemokine binding and signaling through CCR5 remains unknown. We found that cholesterol extraction by beta-cyclodextrin (BCD) significantly reduced the binding and signaling of MIP-1b using CCR5-expressing T cells. Reloading treated cells with cholesterol but not 4-cholesten-3-one, an oxidized form of cholesterol, restored MIP-1b binding to BCD-treated cells. Antibodies specific for distinct CCR5 epitopes lost their ability to bind to the cell surface after cholesterol extraction. Moreover, cells stained with fluorescently-labeled MIP-1b extensively co-localized with the GM1 lipid raft marker while using anti-CCR5 antibodies, the majority of CCR5 on these cells co-localized with CD59 and only partially with GM1 suggesting that active ligand binding facilitates receptor association with lipid rafts or that raft association promotes a higher affinity conformation of CCR5. Together, these data demonstrate that cholesterol and lipid rafts are important for the maintenance of the CCR5 conformation and are necessary for both the binding and function of this chemokine receptor. Similar studies were performed using examining the role of cholesterol in CXCR4 function. We have determined that cholesterol extraction by beta-cyclodextrin (BCD) also inhibits the CXCR4 ligand, SDF-1a, binding to CXCR4 on human T cells. Intracellular calcium responses to SDF-1a, as well as receptor internalization, were similarly impaired in treated T cells. Loss in ligand binding appears to be due to conformational changes in CXCR4 and not increased sensitivity to internalization. SDF-1a binding was effectively restored by reloading cholesterol. These data, along with microscopic evidence that SDF-1a binds within lipid rafts, suggests that cholesterol is essential for CXCR4 function and conformational integrity within lipid rafts.